In this application, Dr. Brian Button is proposing original research to investigate the mechanisms regulating shear stress-mediated ATP release. ATP release and autocrine/paracrine stimulation of purinergic receptors has been implicated in the regulation of a wide array of cell functions in numerous diverse cell types. A key role of purinergic signaling is the regulation of various ion transport processes, including the CFTR chloride channel. External mechanical stresses, including shear, compression, stretch, and cell swelling represent a ubiquitous mechanism to stimulate ATP release. However, the mechanisms responsible for mechanotransduction of external stresses to ATP release are unknown. Recently, the PI and collaborators discovered that the oscillatory nature of stress, such as experienced during normal breathing, is essential to stimulate ATP release. Furthermore, they found that the relationship between the magnitude of oscillatory stress and the rate of ATP release was steepest within the physiological range of normal breathing, whereas stronger forces generated weaker responses. These results lead the PI to hypothesize that cells can actively regulate the rate of ATP release during mechanical stimulation, thus protecting themselves from the potentially detrimental effect of unregulated ATP release and over-stimulation of purinoceptors. Preliminary results suggest that oscillatory stress-mediated ATP release occurs by a mechanism involving transmission of external and cilia beating-mediated forces through the cytoskeleton and exocytosis-dependent secretory pathways. The work outlined in this project is designed to systematically address several components of the mechanotransduction pathway involved in ATP release and establish its physiological role in the regulation of epithelial function. To achieve these objectives, the candidate will employ a variety of techniques grouped into three Specific Aims. Aim 1 will focus on the kinetic properties of stress-stimulated ATP release and identify the cytoskeletal elements involved in the vesicular-mediated process. Aim 2 will test the hypothesis that oscillatory shear stress of magnitude above physiological ranges reduces ATP release by altering the properties of the cell membrane. Finally, Aim 3 will test whether airway epithelia sense and respond to changes in the hydration status of the overlying mucus by internal stresses generated by cilia beating transmitted to the cytoskeleton. Together, these studies are designed to provide invaluable insights into the mechanism regulating ATP release in response to external and internal forces, which may potentially lead to the discovery of novel therapeutic approaches to modulate ATP release, important in such diseases as cystic fibrosis, where ATP release has been shown to stimulate mucus clearance. This K01 award will provide the foundation for Dr. Button to pursue his career goals of becoming an independent investigator and establishing scientific funding opportunities.